Oral kanglaite injection (KLTI) attenuates the lung cancer-promoting effect of high-fat diet (HFD)-induced obesity

Obesity is a risk factor for cancer and cancer-related mortality, however, its role in lung cancer progression remains controversial. This study aimed to assess whether high-fat diet (HFD)-induced obesity promotes lung cancer progression and whether the promotion can be decreased by Kanglaite injection (KLTI). In vivo, HFD-induced overweight or obesity increases the lung carcinoma incidence and multiplicity in a urethane-induced lung carcinogenic model and cancer-related mortality in a LLC allograft model by increasing oxidative stress and cellular signaling molecules including JAK, STAT3, Akt, mTOR, NF-κB and cyclin D1. These changes resulted in increases in vascular disruption and the lung water content, thereby promoting lung epithelial proliferation and the epithelial-mesenchymal transition (EMT) during carcinogenesis. Chronic KLTI treatment substantially prevented the weight gain resulting from HFD consumption, thereby reversing the metabolic dysfunction-related physiological changes and reducing susceptibility to lung carcinogenesis. In vitro, KLTI significantly suppressed the proliferation and induced apoptosis and differentiation in 3T3-L1 preadipocyte cells and attenuated endothelial cell permeability in HUVECs. Our study indicates that there is a potential relationship between obesity and lung cancer. This is the first study to show that obesity can directly accelerate carcinogen-induced lung cancer progression and that KLTI can decrease the lung cancer-promoting effect of HFD-induced obesity.


INTRODUCTION
Lung cancer is the leading cause of cancer death in both more and less developed countries [1], while China is experiencing more and more days of serious air pollution recently and has the highest lung cancer burden in the world [2]. Currently, the control of lung cancer has attracted worldwide attention, whereas focusing on prevention is an important factor to reduce the burden of screening, treatment, and lung cancer deaths [3]. However, much work is needed to determine whether these strategies will be viable. Studies on the relative risk factors of lung cancer have played an important role in its prevention. Recent epidemiological and experimental evidence indicates that obesity and its related metabolic abnormalities are associated with the development of certain epithelial malignancies [4]. There is also expanding evidence of the role of obesity in cancer development, treatment, and survival [5]. Lung cancer is not considered an obesity-related cancer, furthermore, obese patients with non-small cell lung cancer (NSCLC) were reported to have better survival outcomes compared to NSCLC patients of normal weight [6]. However, there is also a possible direct correlation with body mass index (BMI) that would support obesity as a potential risk factor for lung cancer. In fact, cancer patients who are obese are at a greater risk of recurrence with shorter disease-free survival rates than non-obese patients [7]. Although it has been shown enhanced spontaneous metastasis of Lewis lung carcinoma in C57BL/6 mice fed an high fat diet (HFD) [8,9], there is Research Paper no published information regarding whether obesity may affect carcinogen-induced lung cancer.
Kanglaite injection (KLTI) is a microemulsion of Coix seed oil extracted from Chinese medicine-Coix seed which has been used in China as an effective clinical drug for over a thousand years [10]. It contains numerous ingredients (0.1 g Coix seed oil per ml consisting of 20.8% 1,2,3-trioleylglycerol (C 57 H 104 O 6 ), 19.2% 2,3-dioleoyl-1-linoleylglycerol (C 57 H 102 O 6 ), 18.9% 1,2-dilinoleyl-3-oleylglycerol (C 57 H 100 O 6 ), 14.8% 2-linoleyl-3-oleyl-1-palmitoylglycerol (C 55 H 100 O 6 ), 11.8% 2,3-dioleyl-1-palmitoylglycerol (C 55 H 102 O 6 ), 7.5% 1,2-dilinoleyl-3-palmitoylglycerol (C 55 H 98 O 6 ) and 7.0% 1,2,3-trilinoleylglycerol (C 57 H 98 O 6 ), Zhejiang Kanglaite Pharmaceutical Co. Ltd, China), has been proven effective in treating multiple cancers in China, and is also the first drug derived from a traditional Chinese herbal remedy approved by the USA Food and Drug Administration to undergo clinical trials in the United States [11]. A metaanalysis found that KLT injection in combination with chemotherapy was associated with improved response rate, quality of life, and symptoms, and a reduced incidence of adverse events compared with chemotherapy alone in patients with NSCLC [12]. The results from Liu et al also suggest that KLT injection combined with chemotherapy can improve the short-term efficacy, performance status and decrease the risk of gastrointestinal reaction compared with systematic chemotherapy alone in the treatment of advanced non-small cell lung carcinoma [13]. Although some studies have provided observational data on the efficacy of KLTI in cancer treatment, only few of them propose mechanisms that would permit a better understanding of the causal mechanisms of this effect [14]. Considering that Coix seed has been used as a dietary supplement and Coix seed oil could reduce the abdominal fat tissue [15], we hypothesized that HFDinduced obesity facilitates lung carcinoma formation and its pro-carcinogenic effect can be prevented by KLTI. The present experiments were conducted to test that hypothesis in mice fed a HFD or standard rodent chow (SRC) using a urethane-induced lung cancer model and a LLC allograft model, if so, tried to explore its mechanism.

HFD-induced overweight or obesity increases lung carcinoma formation and cancer-related mortality
To assess the potential relationship between obesity and lung cancer, two experimental diets and two lung cancer models were used. Urethane-induced lung cancer has a similar histological appearance and similar molecular changes as those in human lung adenocarcinoma [16]. This animal model has been optimised for manageable carcinogenesis and was used to assess pro-carcinogenic effect of HFD-induced overweight or obesity [17]. LLC allograft model is a suitable mouse model to evaluate therapeutic effects and was used to assess cancer-related mortality [18]. It was shown that the body weight (BW) was 35.5 ± 2.3 g in HFD-fed C57BL/6J mice (overweight, P < 0.05) vs 31.4 ± 2.2 g in SRC-fed C57BL/6J mice and was 56.7 ± 3.1 g in HFD-fed KKAy mice (obesity, P < 0.01) vs 45.6 ± 2.8 g in SRC-fed KKAy mice ( Figure 1A). In addition, in a urethane-induced lung carcinogenesis model, the lung carcinoma incidence was 100% in HFD-fed C57BL/6J mice vs 65% in SRC-fed C57BL/6J mice and was 100% in HFD-fed KKAy mice vs 70% in SRC-fed KKAy mice. Multiplicity was 26.2 ± 4.2 in HFD-fed C57BL/6J mice (P < 0.01) vs 12.3 ± 3.2 in SRC-fed C57BL/6J mice and was 28.7 ± 4.5 in HFD-fed KKAy mice (P < 0.01) vs 13.4 ± 3.3 in SRC-fed KKAy mice ( Figure 1B and 1C). Further, in a LLC allograft model, the tumor size was 3.74 ± 0.43 g in HFD-fed C57BL/6J mice (P < 0.01) vs 2.82 ± 0.41 g in SRC-fed C57BL/6J mice and was 3.85 ± 0.44 g in HFD-fed KKAy mice (P < 0.01) vs 2.91 ± 0.42 g in SRC-fed KKAy mice ( Figure 2A) The number of lung metastasis was 21.3 ± 3.6 in HFD-fed C57BL/6J mice (P < 0.01) vs 12.4 ± 3.3 in SRC-fed C57BL/6J mice and was 24.1 ± 3.8 in HFD-fed KKAy mice (P < 0.01) vs 13.5 ± 3.4 in SRC-fed KKAy mice ( Figure 2B). The median survival of cancer-related survival was 39 d in HFD-fed C57BL/6J mice (P < 0.05) vs 48 d in SRC-fed C57BL/6J mice and was 37 d in HFD-fed KKAy mice (P < 0.05) vs 45 d in SRC-fed KKAy mice ( Figure 2C). However, no significant differences in lung carcinogenesis or tumor development were observed between the two strains of the two lung cancer models following consumption of the same diet ( Figures 1B-1D and 2A-2C), indicating the important role of diet in lung cancer.

HFD-induced overweight or obesity increases lung epithelial proliferation and the EMT
Carcinogenesis is the final result of epithelial proliferation and the epithelial-mesenchymal transition (EMT) [21]. To verify that overweight or obesity plays a role in carcinogenesis, we examined overweight-or obesity-associated changes in lung epithelial proliferation and the EMT by immunohistochemical analysis at 21 week point after induction in a urethane-induced lung carcinogenic model. As shown in Figure 5B-5E, significantly higher levels of the lung epidermal proliferation marker PCNA (score 5 ± 1 in HFD-fed C57BL/6J mice vs 3 ± 1 in SRC-fed C57BL/6J mice (P < 0.01) and 5.5 ± 1 in HFD-fed KKAy mice vs 3 ± 1 in SRC-fed KKAy mice (P < 0.01)), significantly induction of the lung EMT as indicated by the decreased E-cadherin levels (score 1 ± 1 in HFD-fed C57BL/6J mice vs 3 ± 1 in SRC-fed C57BL/6J mice (P < 0.01) and 1 ± 1 in HFD-fed KKAy mice vs 2 ± 1 in SRC-fed KKAy mice (P < 0.05)), and the increased N-cadherin levels (score 5 ± 1 in HFD-fed C57BL/6J mice vs 4 ± 1 in SRC-fed C57BL/6J mice(P < 0.05) and 5.5±1 in HFD-fed KKAy mice vs 4.5 ± 1 in SRC-fed KKAy mice (P < 0.05)) were detected in the overweight and obese mice. These results suggest that overweight and obesity promoted lung epithelial proliferation and the EMT.

KLTI reduces susceptibility to overweight or obesity -promoted lung cancer progression
To confirm whether overweight or obesity is a suitable drug target for KLTI, mice were fed either the SRC or HFD and were orally administered KLTI once daily for 20 weeks. Under these experimental conditions, chronic KLTI treatment did not affect the body weights of the SRC-fed mice (BW 31.4 ± 2.2 g in SRC-fed C57BL/6J mice vs 31.3 ± 2.3 g in SRC+KLTI-fed C57BL/6J mice (P > 0.05) and 45.6 ± 2.8 g in SRC-fed KKAy mice vs 44.5 ± 3.1 g in SRC+KLTI-fed KKAy mice (P > 0.05)). In contrast, this treatment significantly attenuated weight gain in the HFD-fed mice (BW 35.5 ± 2.3 g in HFD-fed C57BL/6J mice vs 32.1 ± 2.4 g in HFD+KLTI-fed C57BL/6J mice (P < 0.05) and 56.7 ± 3.1 g in HFD-fed KKAy mice vs 48.5 ± 3.2 g in HFD+KLTI-fed KKAy mice (P < 0.01)) ( Figure 1A) without affecting the food intake (data not shown). KLTI had a smaller but still significant effect on the lung carcinoma incidence and multiplicity in the two strains of mice fed the SRC compared with those fed the HFD in a urethane-induced lung carcinogenic model ( Figure 1B and 1C, P < 0.05). However, KLTI did not significantly affect tumor size (Figure 2A, P > 0.05), lung metastasis ( Figure 2B, P > 0.05) or cancer-related survival ( Figure 2C, P > 0.05) in the two SRC-fed strains of LLC allograft model mice. In contrast, KLTI significantly decreased tumor development in the two HFD-fed strains of mice, as indicated by the decreases in tumor size (Figure 2A, P < 0.01) and lung metastasis ( Figure 2B, P < 0.01), and increased cancer-related survival ( Figure 2C, P < 0.01).
To clarify the specific role of KLTI in overweightor obesity-related carcinogenesis, growth factor signaling, lung epithelial proliferation, EMT and stem cell activation assays were also performed. Western blot analyses revealed that KLTI decreased cellular signaling molecules including JAK (P < 0.01), STAT3 (P < 0.01), Akt (P < 0.01), mTOR (P < 0.01), NF-κB (P < 0.01) and cyclin D1 (P < 0.01) in the two strains of KLTI-treated mice fed HFD ( Figure 5A). Immunohistochemical analyses demonstrated that the level of the lung epithelial proliferation marker PCNA was significantly reduced ( Figure 5B and 5E, P < 0.01) and that the EMT was significantly suppressed, as indicated by the decreased E-cadherin level ( Figure  5C and 5E, P < 0.01) and the increased N-cadherin level ( Figure 5D and 5E, P <0.01) in the KLTI-treated mice. These results indicate that the KLTI treatment protected mice from HFD-induced overweight and obesity and subsequently reduced their susceptibility to metabolic dysfunction-related lung carcinogenesis.

KLTI induces cell functional changes in vitro
KLTI is a molecularly targeted agent that is prepared as a microemulsion for use in cancer therapy, and it has inhibitory effects on many tumor types [10]. To examine how KLTI affects cell function, we selected Mouse LLC cells which represents lung cancer cells, HUVECs which represents endothelial cell and mouse pre-adipocytes (3T3-L1) which represents adipocytes to conduct assays of cell proliferation, endothelial permeability and differentiation. We found that KLTI at a concentration of less than 200 µg/ml significantly suppressed the proliferation of 3T3-L1 preadipocyte cells in a dose-dependent manner but had little effect on HUVEC and LLC cell proliferation ( Figure 6A). We also found that KLTI treatment prevented the increase in endothelial cell permeability caused by the adipocyte-conditioned media in HUVECs ( Figure 6B). Finally, we examined the effect of KLTI on cell apoptosis and differentiation in 3T3-L1 preadipocytes and found that it increased cell apoptosis at concentrations ranging from 25-100 µg/ml and induced differentiation at concentrations ranging from 6.25-100 µg/ml in 3T3-L1 preadipocytes ( Figure 6C and 6D).

DISCUSSION
Although several potential mechanisms linking obesity and cancers have been proposed [22], no conclusive evidence exists so far with regards to the roles of obesity and its related metabolic abnormalities in lung cancer progression. In this study, we evaluated the effects of HFD-induced overweight or obesity on lung carcinoma initiation and progression using an urethaneinduced lung cancer model and a LLC allograft model. We show that HFD-induced overweight or obesity promoted lung carcinoma formation and progression in these animal models. Moreover, HFD-induced overweight or obesity leads to energy imbalance, decreased levels of cellular signaling molecules and increased lung epithelial (B) PCNA staining, (C) E-cadherin staining, (D) N-cadherin staining., as determined by immunohistochemistry. (E) Immunohistochemical score. Images were captured at ×40 magnification. The data are presented as the mean ± SD of triplicate assays, and statistical significance was determined by ANOVA, and unpaired, 2-tailed Student's t test was used to compare difference between two groups. **vs SRC or ## vs HFD; P < 0.01. HFD, high-fat diet; SRC, standard rodent chow; KLTI, kanglaite injection. Normal was defined as the non-carcinogenic mice fed in parallel. proliferation and the EMT. More importantly, KLTI reduces susceptibility to overweight or obesity-promoted lung cancer progression in vivo and induces cell functional changes in vitro. Here, we not only identified a potential relationship between obesity and lung cancer progression but also provided a strategy for lung cancer prevention.
KLTI, composed of seven types of fatty acids that is extracted from Coix seed, has been demonstrated to be an effective anticancer treatment. Considering that it is from a nutritious food Adlay seed that has no overtly toxic effects and is edible, KLTI was administered by gavage to avoid tumor targeting effect and to assess its preventive effect on overweight or obesity-related pro-carcinogenesis, regardless of drug metabolism. However, the development of preventive agents has unique challenges and special considerations, and it does not differ significantly from Endothelial permeability of HUVECs. (C) 3T3-L1 preadipocyte apoptosis, as determined by AO/EB staining. as (D) 3T3-L1 preadipocyte differentiation, as determined by Oil red staining. The data are presented as the mean ± SD of triplicate assays, and statistical significance was determined by ANOVA, and unpaired, 2-tailed Student's t test was used to compare difference between two groups. *P < 0.05; **P < 0.01 vs KLTI-untreated cells (0 concentration). KLTI, kanglaite injection. the development of therapies for advanced cancers [23]. In this study, we first examined whether overweight or obesity increases the lung carcinoma incidence in a mouse-urethane lung carcinogenic model. We found that HFD-induced overweight in C57BL/6J mice and obesity in KKAy mice significantly increased the lung carcinoma incidence and multiplicity, indicating a potential relationship between obesity and lung cancer. This result was further confirmed in an LLC allograft model, in which overweight and obesity were found to be associated with increased tumor size, to promote lung metastasis and to decrease cancer-related survival. In addition, obesity and overweight increased inflammation and oxidative stress, altered the levels of JAK, STAT3, Akt, mTOR, NF-κB and cyclin D1, and enhanced lung epithelial proliferation and the EMT, thereby demonstrating plausible links between positive energy balance states and lung cancer promotion. Finally, we treated HFD-fed mice with KLTI and found that it significantly decreased lung carcinogenesis and tumor development in the two strains of mice but had reduced or little efficacy in the SRC-fed mice. With regard to results that KLTI did not significantly affect tumor size, lung metastasis or cancer-related survival in the two SRC-fed strains of mice receiving the SRC in the LLC allograft model mice, we believe that the preventive effect of KLTI on overweight and obesity-related pro-cancer is one of its therapeutic characteristic. The therapeutic characteristic of KLTI in adipocytes was further confirmed by the findings that KLTI suppressed cell proliferation and induced cell apoptosis and differentiation in 3T3-L1 preadipocyte cells but had few effects on HUVECs and LLC cell proliferation. Yet, there still remains the possibility that under a non-lethal dosage, KLTI is able to affect the function of HUVECs or LLC cells, such as reduction of endothelial cell permeability and suppression of LLC spontaneous metastasis in this study, which may explain why KLTI still reduced the lung cancer development in urethane-induced lung cancer mice fed the SRC. Simultaneously, it also suggests that KLTI is suitable for obesity-related cancer prevention but not thiner cancer patients.
The relationship between cancer and metabolic disorders was recognized several decades ago, but the underlying mechanisms involved in cancer development and progression remain obscure [24]. We has revealed that tissue interstitial fluid (TIF) provides better nutrition to tumors than angiogenesis and that it promotes the development of malignant phenotypes of lung cancer independently of angiogenesis [25]. In this study, we found that overweight or obesity decreased lung epithelial integrity, promoted vascular disruption and increased the lung water content. Considering our finding that KLTI did not significantly affect on tumor size, lung metastasis or cancer-related survival in the two SRC-fed strains of LLC allograft model mice and the fact that KLTI alone cannot cure lethal human malignancies [13], we believe that the pro-TIF effect of overweight and obesity is one of the main mechanisms underlying the increase in cancerrelated mortality, and we support the use of Coix seed as a candidate strategy for preventing obesity-related carcinogenesis and cancer edema-related mortality.
An understanding of the interplay between local and systemic alterations involved in the obesity-cancer link provides the basis for developing interventions aimed at mitigating the protumorigenic effects [26]. Most studies support a relationship between body adiposity and sitespecific mortality or cancer progression [27]. However, most of the research was not specifically designed to study these outcomes and, therefore, several methodological issues should be considered before integrating their results to draw conclusions [28]. Our results provide new insights into the role of HFD-induced obesity in lung cancer and demonstrate that overweight and obesity can increase lung cancer formation. This is the first study to show that obesity can directly accelerate carcinogen-induced lung cancer progression and oral KLTI can decrease the procarcinogenic effect of HFD-induced overweight or obesity. Many researchers have proposed the concept of "survival with cancer" and insist that controlling cancer and causing cancer cells to "remain static" and to "hibernate" for a long period time is better than attempting to reduce the tumor volume or completely kill all cancer cells [29]. Based on this view, we believe that KLTI Coix seed oil possesses advantages as an adjuvant therapy for alleviating the symptoms of terminal-stage cancer, for which no further treatment options can be offered in Western medicine.

Animals
Cohorts of 5-to 6-week old female C57BL/6J mice and KKAy mice were obtained from Beijing Weitong Lihua Animal Co. All mice were housed in individual ventilated cages under a 12-h light-dark cycle (lights on from 7:00 AM to 7:00 PM). The animals were fed aSRC (Henan Provincial Medical Laboratory Animal Center) or a HFD ( Beijing Weitong Lihua Animal Co.) and water. All animal procedures used in this study were approved by the Animal Experimentation Ethics Committee (AEEC) of Henan University (permission number HUSAM2014-216), and all procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals and the regulations of the animal protection committee to minimize suffering and injury. The animals were monitored daily and euthanized humanely by an overdose of carbon dioxide (CO 2 ) at the end of the experiment or at the first sign of shortness of breath, reduced locomotion and reduced body weight (greater than 20% of the total body weight). All surgeries were performed under general anesthesia ( intraperitoneal injection of 45 mg/kg pentobarbital sodium, R&D Systems, M→N, USA), and all efforts were taken to minimize suffering.

Urethane-induced lung adenocarcinoma model
C57BL/6J or KKAy mice were administered freshly prepared urethane to induce lung adenocarcinoma according to our previously published protocol [17]. The mice received intraperitoneal injection of urethane (U2500, Sigma, St. Louis, MO, USA) (600 mg/kg body weight, dissolved in sterile 0.9% NaCl) once weekly for 10 weeks. Following urethane injection, the mice were fed the SRC or HFD (60% kcal from fat) and received 20 ml/kg KLTI (containing 0.1 g Coix seed oil per ml for injection) by gavage once daily for 20 weeks. During the study, food and water were provided ad libitum. The health of the mice was monitored daily, and their body weights were measured weekly. At 21 weeks after the first urethane injection, orbital venous blood was collected for use in a plasma 5-HIAA (a vascular disruption marker) assay performed using an ELISA kit (R&D Systems, MN, USA). Finally, the mice were euthanized, the lung carcinoma incidence and multiplicity (average number of carcinomas per mouse) were determined, and the lungs were biochemically processed.
Each group contained twenty-two mice in experiments, and a normal group was included that contained non-cancerous mice fed in parallel. At the end of the experiment, each group contained at least twenty mice. In all experimental procedures, one to two mice in each group exhibited clear evidence of myocardial infarction and were excluded from the study and humanely euthanized.

Insulin and glucose tolerance tests
Two tests were carried out at 1 week before the mice were euthanized. For the insulin tolerance test, the mice were fasted for 6 h and were then intraperitoneally injected with 1 U/kg BW insulin (Sigma, St. Louis, MO). For the glucose tolerance test, the mice were fasted for 16 h and were then intraperitoneally injected with 2 g/ kg BW glucose (Sigma, St. Louis, MO, USA). Blood was collected from the tail veins of the mice at 0, 15, 30, 60, and 90 min after injection, and the glucose concentrations were analyzed using a Glucometer (Bayer, Pittsburgh, PA). Oxidative stress analysis 8-OHdG (R&D Systems, MN, USA) levels were measured using an ELISA assay kit. Serum levels of ROS (Nanjing Jiancheng Bioengineering Institute, China), a marker of oxidative stress, were also determined using an ELISA assay kit.

Lung epithelial integrity and lung water content measurements
The integrity of the lung epithelium was assayed using the Evans blue extra-barrier technique as previously described [29], and lung water content was measured as previously described [25].

Immunohistochemistry
For immunohistochemical (IHC) analyses, serial lung sections (5 μm) were cut and attached to Superfrost Plus microscope slides. The slides were warmed to 60°C for 1 h, deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. After being blocked with 3% hydrogen peroxide for 10 min, the sections were incubated with 0.1% Triton X-100 in PBS for 20 min at room temperature. Then, they were immersed in a blocking solution containing 5% BSA for 20 min at room temperature. Next, the sections were incubated with primary antibodies (1:100) against PCNA (ProteinTech Group, Wuhan, China), E-cadherin (ProteinTech Group, Wuhan, China) and N-cadherin (ProteinTech Group, Wuhan, China) overnight at 4°C. Antigen-antibody binding was detected using a Dako Cytomation LSAB System-HRP Kit (Dako Cytomation, Carpinteria, CA, USA). Signal was developed using the peroxidase substrate DAB (Nanjing Jiancheng Bioengineering Institute, Chinawhich, yields a brown reaction product) or using AEC (Nanjing Jiancheng Bioengineering Institute, China, yields a red reaction product). All sections were counterstained with hematoxylin and imaged under a microscope. Semiquantitative histological scores (HSCORE) wwere calculated, as described previously [29].

Western blot analysis
Lung extracts were prepared in radioimmunoprecipitation assay (RIPA) cell lysis buffer (Solarbio, Beijing, China), and equal amounts of protein were separated on a 12% sodium dodecyl sulfatepolyacrylamide electrophoresis gel, electroblotted on nitrocellulose membranes, and probed with specific . The secondary antibody used was Peroxidase-conjugated Affinipure Goat anti-rabbit IgG (H + L) (ProteinTech Group, 1:5000). Antibody binding was detected by enhanced chemiluminescence according to the manufacturer's protocol (Pierce, Rockford, IL). Band densities were quantified using Image software (NIH, Bethesda, MD, USA) and normalized according to the reference gene GAPDH. All values were then normalized to relative GAPDH expression.

Tumor allograft experiment
LLC cells were maintained in RPMI 1640 medium containing 10% FBS until they reached 80% confluence. Then, the cells were collected, and 200 µL saline containing 1 × 10 6 LLC cells was injected subcutaneously into the lateral axilla of normal C57BL/6J mice or KKAy mice, as well as into those of HFD-fed lean C57BL/6J mice or obese KKAy mice. Following tumor inoculation, the mice were administered 20 ml/kg KLTI by gavage once daily for four weeks. At 28 days after tumor incubation, the tumors were dissected and weighed, and the number of tumors on the lung surface was counted.
Ten mice in each group were used for survival analysis until all of the mice died.

Cell proliferation assay
3T3-L1 preadipocyte cells and HUVECs were seeded at 10,000 cells per well and LLC cells were seeded at 5,000 cells per well in 96-well plates overnight at 37°C. Cells were treated with the indicated concentrations of KLTI, and each condition was tested using six replicates. During the final 4 h of the 48-h incubation period, the supernatants were discarded, and 100 μL MTT (Solarbio, Beijing, China) (0.5 mg/ml) was added to each well. After 4 h, the MTT was discarded, and 100 μL DMSO (Solarbio, Beijing, China) was added to each well. The absorbance of each sample was measured at 570 nm using a plate reader (Elx-800; Bio-Tek, USA). The results of the cell proliferation assay are presented as a percentage of the control cells. All experiments were repeated in triplicate.

Adipocyte differentiation
3T3-L1 preadipocyte cells were cultured in DMEM complete medium and grown to confluence. The medium was replaced with complete medium containing 1.7 µM insulin, 1.0 µM dexamethasone and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Solarbio, Beijing, China), and the cells were cultured for 48 h. Then, the medium was replaced with complete medium containing 1.7 µM insulin (Sigma, St. Louis, MO, USA) and cultured for an additional 48 h. Finally, the cells were maintained in complete medium containing the indicated concentrations of KLTI as Coix seed oil, and the medium was replaced with fresh medium containing the indicated concentrations of KLTI every 48 h. On day 8, the supernatant was collected and filtered to remove debris to obtain adipocyteconditioned medium. The cells were washed twice with cold PBS (136.9 mM NaCl, 2.68 mM KCl,4 mM Na2HPO4, and 1.76 mM KH2PO4; pH 7.4), fixed in 3.7% paraformaldehyde (Solarbio, Beijing, China) for 1 h and then stained with 0.2% (w/v) Oil red O(Rowley Biochemical, USA) in a 60% isopropanol (IT7048, Shanghai Sangon Biological Engineering Co. Ltd, China) solution for 1 h. Oil red staining was observed and analyzed using an inverted microscope (Olympus, Tokyo, Japan). Stained cells were treated with 100% isopropyl alcohol to extract the Oil red O, and the colorimetric intensity of the extract was measured at 595 nm to determine the lipid content.

In vitro permeability assay
Endothelial cell permeability was quantified by spectrophotometric measurement of the flux of Evans bluebound albumin (Solarbio, Beijing, China) across functional cell monolayers using a modified two-compartment chamber model. Briefly, HUVECs were plated (5 × 10 4 cells/well) in a 3-μm-pore size, 12-mm-diameter Transwell apparatus for 3 days. Confluent monolayers were incubated in medium supplemented with 10% adipocyte-conditioned medium for 24 h. Then, the transwell inserts were washed with PBS, and 0.5 ml Evans blue (0.67 mg/ml) diluted in growth medium containing 4% bovine serum albumin (BSA) (Life Technologies, New York, USA) was added. Fresh growth medium was added to the lower chamber, and the medium in the upper chamber was replaced with Evans blue/BSA. Ten minutes later, the optical density of the solution in the lower chamber was measured at 650 nm.

Apoptosis detection
Apoptosis was evaluated by acridine orange (AO) / ethidium bromide (EB) (Sigma, St. Louis, MO, USA) fluorescence staining. Briefly, 4 μL of dye mixture (100 μg/mL AO and 100 μg/mL EB in PBS) were added to a suspension of KLTI-treated LLC cells cultured under serum-free conditions for 12 h, and the cells were washed once with PBS. After staining, the cells were visualized and analyzed using an IN Cell Analyzer 2000 (GE Healthcare).

Statistical analyses
The Shapiro-Wilk test was conducted to test for normal distribution. Normally distributed data are presented as the mean ± SD and were statistically analyzed using GraphPad Prism, Version 5.0 (San Diego, CA, USA). Differences among the groups were evaluated by ANOVA one-way or two-way analysis. A P value of less than 0.05 was considered statistically significant.